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The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

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Population modelling of vasa-driven HEGs targeting female-specific genes. (A) and (B) HEG constructs targeting a somatic female gene for invasive vector control, (C) HEG constructs targeting a germline-specific female gene for non-invasive vector control. A) The threshold rate of homologous repair in the germline (hg) needed for the HEG to invade a population from low frequency, as a function of the overall rate of cleavage. Invasiveness of such a construct is defined as its ability to spread from rare through a population by virtue of genetic drive. The threshold differs depending upon the rate of homing in the embryo (he) (red vs. blue lines). Conversely, with no maternal deposition, the HEG will invade for any hg>0. (B) Frequency of the HEG and population mean fitness assuming c = 0.9, hg = 0.9, and an initial release frequency of 1%. Black line: no maternal deposition (D = 0); red line: maternal deposition in which homing rates in the embryo mirror those in the germline (D = 1; he = hg); blue line: maternal deposition in which all cut sites are repaired exclusively by non-homologous repair (D = 1; he = 0). (C) Number of sterile females produced per released male for the HEG-based non-invasive strategy proposed, compared to classical inundative control strategies. To be able to compare to classical measures constructs are released in homozygote males assuming c = 0.9 and for simplicity the models assume that on average one male mates once in his lifetime. c rate of cleavage); h (rate of homologous repair); D (maternal deposition).
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Figure 5: Population modelling of vasa-driven HEGs targeting female-specific genes. (A) and (B) HEG constructs targeting a somatic female gene for invasive vector control, (C) HEG constructs targeting a germline-specific female gene for non-invasive vector control. A) The threshold rate of homologous repair in the germline (hg) needed for the HEG to invade a population from low frequency, as a function of the overall rate of cleavage. Invasiveness of such a construct is defined as its ability to spread from rare through a population by virtue of genetic drive. The threshold differs depending upon the rate of homing in the embryo (he) (red vs. blue lines). Conversely, with no maternal deposition, the HEG will invade for any hg>0. (B) Frequency of the HEG and population mean fitness assuming c = 0.9, hg = 0.9, and an initial release frequency of 1%. Black line: no maternal deposition (D = 0); red line: maternal deposition in which homing rates in the embryo mirror those in the germline (D = 1; he = hg); blue line: maternal deposition in which all cut sites are repaired exclusively by non-homologous repair (D = 1; he = 0). (C) Number of sterile females produced per released male for the HEG-based non-invasive strategy proposed, compared to classical inundative control strategies. To be able to compare to classical measures constructs are released in homozygote males assuming c = 0.9 and for simplicity the models assume that on average one male mates once in his lifetime. c rate of cleavage); h (rate of homologous repair); D (maternal deposition).

Mentions: Based on the properties of the Vas2GFP construct we set out to investigate how the expression characteristics of the A. gambiae vasa regulatory region would affect its performance in potential vector control strategies. In particular, we were interested to determine how maternal deposition would affect the effectiveness of HEG-based control methods that aim to reduce mosquito population numbers by imposing a genetic load. Previous models have considered a HEG designed to target a somatically expressed gene essential for female viability or fertility, whose knockout is recessive but has no effect on male fitness [12]. The models predicted that if such a HEG is released at low frequencies into a population it would invade and reach an equilibrium frequency. The maximum equilibrium frequency achievable would depend upon the efficiency of target cleavage and the relative frequencies of homologous repair (HR) and non-homologous repair (NHR) [12,34]. In our model, by virtue of maternal deposition, the homing endonuclease is also active against the zygotic genome of embryos originating from females carrying the HEG allele. Homozygous mutant offspring, inviable or sterile in the case of females, can thus arise by inactivation of the target genes via HR or NHR in embryos even if the paternally derived allele is originally wild-type. Since maternal deposition of the homing endonuclease can reduce the reproductive fitness of heterozygote females, because their daughters are inviable or sterile, the HEG will not necessarily spread from rare, but instead will only invade the population if the rate of repair by homologous recombination is above a threshold value in reference to a particular rate of cleavage (Figure 5A). Moreover, the equilibrium frequency of a HEG that is maternally deposited will significantly depend on the outcome of HEG-induced cleavage in the embryo. HEG equilibrium frequencies remain unaffected, when the relative rate of HR in embryos mirrors that of the germline (he = hg), but when all cut sites in embryos are repaired by NHR (he = 0) then the equilibrium frequency is lowered (Figure 5B). The genetic load imposed by a maternally deposited HEG can be higher, due to the extra lethality or sterility. Reduced population fitness can therefore be achieved, although this would require HR in embryos (Figure 5B).


The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Population modelling of vasa-driven HEGs targeting female-specific genes. (A) and (B) HEG constructs targeting a somatic female gene for invasive vector control, (C) HEG constructs targeting a germline-specific female gene for non-invasive vector control. A) The threshold rate of homologous repair in the germline (hg) needed for the HEG to invade a population from low frequency, as a function of the overall rate of cleavage. Invasiveness of such a construct is defined as its ability to spread from rare through a population by virtue of genetic drive. The threshold differs depending upon the rate of homing in the embryo (he) (red vs. blue lines). Conversely, with no maternal deposition, the HEG will invade for any hg>0. (B) Frequency of the HEG and population mean fitness assuming c = 0.9, hg = 0.9, and an initial release frequency of 1%. Black line: no maternal deposition (D = 0); red line: maternal deposition in which homing rates in the embryo mirror those in the germline (D = 1; he = hg); blue line: maternal deposition in which all cut sites are repaired exclusively by non-homologous repair (D = 1; he = 0). (C) Number of sterile females produced per released male for the HEG-based non-invasive strategy proposed, compared to classical inundative control strategies. To be able to compare to classical measures constructs are released in homozygote males assuming c = 0.9 and for simplicity the models assume that on average one male mates once in his lifetime. c rate of cleavage); h (rate of homologous repair); D (maternal deposition).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713240&req=5

Figure 5: Population modelling of vasa-driven HEGs targeting female-specific genes. (A) and (B) HEG constructs targeting a somatic female gene for invasive vector control, (C) HEG constructs targeting a germline-specific female gene for non-invasive vector control. A) The threshold rate of homologous repair in the germline (hg) needed for the HEG to invade a population from low frequency, as a function of the overall rate of cleavage. Invasiveness of such a construct is defined as its ability to spread from rare through a population by virtue of genetic drive. The threshold differs depending upon the rate of homing in the embryo (he) (red vs. blue lines). Conversely, with no maternal deposition, the HEG will invade for any hg>0. (B) Frequency of the HEG and population mean fitness assuming c = 0.9, hg = 0.9, and an initial release frequency of 1%. Black line: no maternal deposition (D = 0); red line: maternal deposition in which homing rates in the embryo mirror those in the germline (D = 1; he = hg); blue line: maternal deposition in which all cut sites are repaired exclusively by non-homologous repair (D = 1; he = 0). (C) Number of sterile females produced per released male for the HEG-based non-invasive strategy proposed, compared to classical inundative control strategies. To be able to compare to classical measures constructs are released in homozygote males assuming c = 0.9 and for simplicity the models assume that on average one male mates once in his lifetime. c rate of cleavage); h (rate of homologous repair); D (maternal deposition).
Mentions: Based on the properties of the Vas2GFP construct we set out to investigate how the expression characteristics of the A. gambiae vasa regulatory region would affect its performance in potential vector control strategies. In particular, we were interested to determine how maternal deposition would affect the effectiveness of HEG-based control methods that aim to reduce mosquito population numbers by imposing a genetic load. Previous models have considered a HEG designed to target a somatically expressed gene essential for female viability or fertility, whose knockout is recessive but has no effect on male fitness [12]. The models predicted that if such a HEG is released at low frequencies into a population it would invade and reach an equilibrium frequency. The maximum equilibrium frequency achievable would depend upon the efficiency of target cleavage and the relative frequencies of homologous repair (HR) and non-homologous repair (NHR) [12,34]. In our model, by virtue of maternal deposition, the homing endonuclease is also active against the zygotic genome of embryos originating from females carrying the HEG allele. Homozygous mutant offspring, inviable or sterile in the case of females, can thus arise by inactivation of the target genes via HR or NHR in embryos even if the paternally derived allele is originally wild-type. Since maternal deposition of the homing endonuclease can reduce the reproductive fitness of heterozygote females, because their daughters are inviable or sterile, the HEG will not necessarily spread from rare, but instead will only invade the population if the rate of repair by homologous recombination is above a threshold value in reference to a particular rate of cleavage (Figure 5A). Moreover, the equilibrium frequency of a HEG that is maternally deposited will significantly depend on the outcome of HEG-induced cleavage in the embryo. HEG equilibrium frequencies remain unaffected, when the relative rate of HR in embryos mirrors that of the germline (he = hg), but when all cut sites in embryos are repaired by NHR (he = 0) then the equilibrium frequency is lowered (Figure 5B). The genetic load imposed by a maternally deposited HEG can be higher, due to the extra lethality or sterility. Reduced population fitness can therefore be achieved, although this would require HR in embryos (Figure 5B).

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

Show MeSH
Related in: MedlinePlus